Automatic tissue processor

ABSTRACT

A tissue processor is programmed for fixation, dehydration and clearing of electron microscopy size particles and utilizes a porous receptacle for each particle or group of particles of tissue and a processing chamber adapted to contain a plurality of the receptacles. The chamber is connected to a plurality of containers, some of which are refrigerated, and which contain the various processing solutions. The solutions are individually piped to, and from beneath, the chamber through a remotely controlled valve and manifold arrangement which also connects with a metering pump. This arrangement minimizes fluid contamination and allows each solution to be precisely metered, brought to, retained in and drained from the chamber thus allowing the particles to be bathed in the solutions according to an automatic programmed time sequence. The program may be varied as to number of cycles per solution, as to number of solutions per program, as to time per cycle and as to starting and terminal solutions in the program.

United States Patent Kinney et al.

[ AUTOMATIC TISSUE PROCESSOR [76] lnventors: Thomas D. Kinney, 3120Devon Rd.; John E. P. Pickett, 3323 Pinafore Dr., both of Durham, NC.

22 Filed: Feb. 22, 1972 [21] Appl. N0.: 228,115

[52] U.S. Cl ..1l8/7,118/421, 222/136 [51] Int. Cl. B05c 11/10 [58]Field of Search 118/4, 7, 8, 421, 118/429, 50; 134/96, 97, 98, 195;137/1 NE; 222/136, 144.5, 144, 190; 95/93 [56] References Cited UNITEDSTATES PATENTS 2,774,364 12/1956 Brobeil 222/136 X 2,959,151 1l/1960Ehrlich 118/429 3,092,121 6/1963 Broge 134/97 X 3,227,130 1/1966Weiskopf 118/11 3,294,101 12/1966 Suzuki et al.. 134/95 X 3,392,7806/1968 Brown 118/429 3,400,726 9/1968 DuGrail 134/95 3,452,710 7/1969l-lentschel 118/7 3,526,203 9/1970 Kinney 118/7 3,550,552 12/1970Archibald 1123/? O WASTE Primary ExaminerMorris Kaplan AttorneyB. B.Olive [57] ABSTRACT A tissue processor is programmed for fixation,dehydration and clearing of electron microscopy size particles andutilizes a porous receptacle for each particle or group of particles oftissue and a processing chamber adapted to contain a plurality of thereceptacles. The chamber is connected to a plurality of containers, someof which are refrigerated, and which contain the various processingsolutions. The solutions are individually piped to, and from beneath,the chamber through a remotely controlled valve and manifold arrangementwhich also connects with a metering pump. This arrangement minimizesfluid contamination and allows each solution to be precisely metered,brought to, retained in and drained from the chamber thus allowing theparticles to be bathed in the solutions according to an automaticprogrammed time sequence. The program may be varied as to number ofcycles per solution, as to number of solutions per program, as to timeper cycle and as to starting and terminal solutions in the program.

18 Claims, 11 Drawing Figures PATENTEmmv I 3 I975 3771,2190

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TO WASTE AUTOMATIC TISSUE PROCESSOR CROSS REFERENCE TO RELATEDAPPLICATIONS This application is related to copending applications Ser.No. 860,256, filed Sept. 23, 1969, now U.S. Pat. No. 3,697,299 entitledMicroscopy Tissue Receptacle Method and Ser. No. 60,798, filed Aug. 4,1970, now abandoned entitled Electron Microscopy Tissue ProcessingMethod.

BACKGROUND or THE-INVENTION 1. Field of the Invention This inventionrelates broadly to apparatus and devices for processing pieces of tissuebut particularly to apparatus for processing electron microscopy sizetissue.

2. Description of the Prior Art This invention constitutes animprovement upon the subject matter of our prior U.S. Pat. No.3,526,203, upon the subject copending applications and upon the methodsrelated thereto. Since the general history of the prior art is dealtwith in some detail in prior U.S. Pat. No. 3,526,203 reference is madeto U.S. Pat. No. 3,526,203 for the prior art background of the presentinvention. Reference is also made to prior U.S. Pat. No. 3,536,040directed to a selectively permeable receptacle and to U.S. Pat. No.3,576,176 directed to a tissue capsule rack which forms part of theprior art of specific interest and useful in practicing the presentinvention. In summary, the subject U.S. Pat. No. 3,526,203 discloses aprocessor for processing electron microscopy size tissue, as well aslarger tissue, in a permeable receptacle of the type disclosed in U.S.Pat. No. 3,536,040 with the receptacle being held in a rack as shown inU.S. Pat. No. 3,576,176. Our own prior art is believed to be the closestknown prior art. Of particular interest to the present invention is thatthe processor of our prior patent disclosed a system of connecting theprocessing chamber with the processing solution containers by means ofpipes which connected to the top of the processing chamber and dependedon gravity feed and further depended on sensing the level of thesolution in the chamber as a means of stopping flow and measuring thevolume of a particular solution. After extensive experimental use it wasfound that the processor of our prior patent inherently introducedsubstantial opportunity for the solutions to mix in the various pipesbetween the chamber and the containers. Also, after experimental usewith various types of solutions, particularly osmic acid, and timeprograms the prior processor was found to introduce a problem inmaintaining accurate level control for the various solutions. Certainsolutions when passed through relatively long lines tended toprecipitate in the lines particularly when the solution was held ator=above room temperature and no provision was made in the priorprocessor for maintaining selected solutions at temperatures below roomtemperature.

Of particular interest is the fact that while our prior patent taughtthe general concept of automatic operation with a program switch theprocessor of the prior patent did not disclose means for convenientlychanging the numbers of cycles for a particular solution. That is, inhand processing it is sometimes the practice to hold the tissue in asolution, then drain the solution, then hold the tissue again in thesame solution for another time period. The processor of our prior patentdid not provide a means for duplicating in a machine operation thisaspect of hand processing. While the prior patent processor contemplatedchanging an entire program in order to obtain such a variation innumbers of cycles per solution the processor of our prior patent did notnot offer the possiblity of making such a change even while a particularprogram was in progress. Furthermore, the program capability of theprocessor of our prior patent was to some extent limited in being ableto vary the amount of time devoted to a particular solution being in theprocessing chamber. Thus, with the processor and processing method ofour prior patent if it was desired to change the base time during whicha particular solution was held in the processing chamber from, say, 12minutes to 24 minutes it was necessary to change the entire program forthe entire run of solutions. Also, the prior processor did not exhibitthe capability of being able to pass certain solutions in a program orto terminate a particular program, at least temporarily, with aparticular solution. In hand processing of electron microscopy tissue itis sometimes desirable to, say, stain a tissue after processingsolutions 1, 2 and 3 and to, say, bypass solution 4 with a certaintissue.

From the foregoing it can thus be seen that while the processor of ourprior patent constituted an important advance in the art and so far aswe know constituted the first significant step toward a practicalautomatic processor for processing electron microscopy size tissue longand extensive experimental use with that processor have revealed anumber of needed improvements in terms of both apparatus and suchconstitutes the object of the present invention.

SUMMARY OF THE INVENTION The invention is generally directed to anapparatus for processing electron microscopy size tissue and constitutesan improvement over the apparatus shown called for in our prior U.S.Pat. No. 3,536,203. Reference to our prior patent is made for anunderstanding of the general operation involved with an apparatus ofthis kind. Of specific interest to the present invention, the apparatusembodies a stationary processing chamber having a bottom port connecteddirectly to a manifold which is positioned below the chamber. Themanifold is also provided with a plurality of inlets connected throughpipes to each of the respective processing solutions and with an outletfor waste discharge. Each inlet on the manifold mounts a remotelysolenoid controlled valve so that the solutions from each solutioncontainer can be effectively isolated from each other at pointsimmediately adjacent the manifold which minimizes the opportunity for,say, solution 1 to mix with solution 3 and also minimizes the amount ofnecessary waste solution. The chamber port communicates with a solenoidcontrol valve immediately adjacent the manifold as does the wasteoutlet. A metering pump also connects to the manifold and incorporates ametering piston driven by a reversible motor so that the piston can bewithdrawn to pull in a given amount of solution and can be moved in theopposite direction to force the solution into the processing chamberthrough the mentioned bottom port. The manifold thus provides a passagefor a selected solution to be withdrawn into the pump and to then beforced into the processing chamber. During withdrawal all valves exceptthe selected solution valve are closed. On the return stroke only theprocessing chamber valve is opened and during dumping only the wastevalve is opened to allow the expended solution to drain by gravity.Means are provided whereby the amount of pump cylinder movement can beprecisely regulated to vary the amount of solution withdrawn so as toeliminate the need for level sensing and control as in the processor ofour prior patent. The various solenoid valves are also coordinated byprogram switch means such that all the respective solutions may bemetered, pumped up to the processing chamber and then allowed to drainby gravity to waste with minimal mixing of one solution with anotherexcept in the collective waste.

For greater variety of programming, the invention also adopts theconcept of establishing a so-called base time of, say, 15 minutes andincorporating means whereby a particular solution may be held during aparticular cycle for one, two or three periods of base time. Thus,merely by moving a switching arrangement asolution can be held for, say,15, 30 or 45 minutes. This can be further varied so that, during aparticular program, solution 1 stays for, say, 15 minutes, solution 2for 45 minutes and solution 3 for 45 minutes in that sequence. Theprogram can be changed while in progress or from program to programmerely by reorienting a set of switching positions. Thus, a medicalresearch investigator is offered a wide range of processing techniquesfor comparing one mode of processing the same tissue or different tissueparticules with another mode of processing. The processor of theinvention also mounts a switching arrangement adapted to allow anyparticular solution to be bypassed in a program or for the processing tostop, at least temporarily, when such solution is reached. An improvedventilation system has also been provided which allows -fumes in thework area and particularly around the processing chamber to be withdrawnas rapidly as formed.

In summarizing the present invention in apparatus terms, it can be saidthat the programming capability has been substantially increased, theopportunity for solution mixing has been minimized and control over thequantity of solution used has been made much more precise. In methodterms, it can also be said that the method of the invention minimizesintermixing of solutions, increases accuracy of measuring and providesgreater variety in programming. Other improvements of an important butlesser nature will also be noted as the description proceeds.

DESCRIPTION OF THE DRAWINGS FIG. 1 is a perspective view of an apparatusfor processing electron microscopy tissue according to the invention.

FIG. 2 is a right elevation view of the apparatus.

FIG. 3 is a rear elevation view of the apparatus.

FIG. 4 is a left elevation view of the apparatus with variouscompartment doors open.

FIG. 5 is an enlarged front elevation view of the main control panel andalso showing the auxiliary control panel in an open position.

FIG. 6 is a front elevation view of the solution containers with thecontainer compartment doors being shown in an open position.

FIG. 7 is a perspective somewhat schematic view of the manifold,metering pump, processing chamber and waste tank arrangement.

FIG. 8 is a sectional and somewhat schematic elevation view of thechamber, valveand metering pump arrangement.

FIG. 9 is a plan, sectional and somewhat schematic view of the manifold,passages, valves and metering pump.

FIG. 10 is a schematic diagram of the control system.

FIG. 11 is a perspective view of a prior art porous receptacle used withthe invention.

DESCRIPTION OF THE PREFERRED EMBODIMENT The present invention isdirected to processing apparatus and a method for processing tissueparticles held in a porous receptacle 25 of the type shown in FIG. 11and more fully disclosed in related US. Pat. No. 3,536,040. A typicalreceptacle of this type is porous at least in some portion, whether inthe body 20 or the end caps 21, to all the solutions. It may be roughlyonehalf inch in length and one-half inch in diameter and the pore sizein the range of about 40 to 200 microns. A substantial number of suchreceptacles may be processed simultaneously and according to a timeschedule for the various processing solutions as later discussed.

The processor of the invention includes a cabinet 29 having pivotedfront doors 30, 31, a pivoted rear door 32 and a pivoted top door 33.Doors 30, 31 cover, respectively, a refrigerated compartment 40 and anonrefrigerated compartment 41 having a dividing partition 42 and a backwall partition 43. Suitable heat insulation 45 is mounted on door 31 andsurrounding compartment 40 in order to retain the reduced temperatureobtained by suitable refrigeration mechanism generally indicated at 46.Ten solution containers 50-59 are shown by way of example and arelabeled respectively: solution 1, 4 percent glutaraldehyde; solution 2,buffer; solution 3, 1 percent osmium tetroxide; solution 4, 50 percentalcohol; solution 5, percent alcohol; solution 6, 80 percent alcohol;solution 7, 95 percent alcohol; solution 8, absolute alcohol; solution9, propylene oxide; solution 10, propylene oxide-Epon 50-50. The firstthree mentioned solutions have been found to require refrigeration forbest processing results and it is for this reason that compartment 40 isrefrigerated. Each solution container is provided with a suitablequick-disconnect 70, FIG. 6, and an air inlet 71 to allow the solutionto be withdrawn through a connecting tube 72. The respective connectingtubes 72 are each connected to a respective solenoid valve 73 which inturn are mounted on a manifold structure 75. Structure 75, as shown inFIG. 9, provides a series of short passages for communicating all of thevarious solution, chamber, waste and pump inlets and ports. Like othercomponents exposed to the solutions, structure 75 must be of a materialinert to all the solutions and stainless steel and Teflon plastic havebeen employed. The ten tubes 72 leading from the ten solution containers50-59 are connected to ten respective solenoid valves 73. Manifold 75also mounts above the manifold a solenoid valve having a connecting pipe81 connected to the processing chamber 90 and a pipe 82 connected to themanifold 75. Below the manifold there is mounted a further solenoidvalve 85 having a pipe 86 connecting to the manifold 75 and a pipe 87connecting to a waste tank 88. The exposed portions of the valves mustbe inert and are preferably stainless steel with Teflon inserts thoughother materials may be employed if suitably inert to the solutions.

Manifold is also connected through a pipe with a pump metering chambergenerally indicated at 101. All parts of the metering pump exposed tothe solution should be relatively inert. Teflon plastic and stainlesssteel have been employed for this purpose. Chamber 101 is formed in apiston operated metering pump which is designated 99 and in this caseincludes a reversible electric motor 105, FIG. 7, connected to drive aninternally threaded tube member 106 which in turn receives a threadedpiston rod 107 such that when the armature of motor rotates in onedirection piston rod 107 is moved forwardly to move piston 110 towardthe cylinder port 111 and when the motor armature turns in the oppositedirection piston 110 is moved away from port 111. Piston rod 107 mountsa pin 112 which moves back and forth in a slot 113 located in bracket114. Four micro-switches 115, 116, 117 and 118 are mounted in the pathof retracting movement of pin 112 so as to strike pin 112 and beactuated. Switches 115, 116, 117 and 118 are arranged in the circuitryso that one of the switches can be selected to control the amount ofmovement of pin 112 during retraction by deenergizing motor 105 and bythis means very precisely control the amount of solution metered by pump99 and indicated by levels A, B, C and D. It should be understood thatappropriate circuitry commands motor 105 to turn to advance piston 110when a solution is to be metered, other circuitry commands piston 110 toretract, other circuitry selectively controlled by micro-switches116-118 command motor 105 to stop and other circuitry commands motor 105to turn so as to advance piston 110 to pump out the metered solution.Annular piston edges 109 are formed to assist sealing.

The processing chamber 90 of the present invention is generally like butdiffers in an important respect from that shown in prior US. Pat. Nos.3,526,203 and 3,576,l76. Specifically, the solutions, instead of beingadmitted at the top through plural inlets, are both admitted anddischarged through a single port 91 in the bottom of chamber 90. Levelsensing is thus avoided by means of the described metering pump. Chamber90 has a tapered bottom which assists in dumping solutions and alsoallows a relatively small number of capsules to be processed. Todescribe the processing chamber 90 in more detail it will be seen thatthe removable cap 120 is provided with a clear glass window 121. Chamber90 is physically located within cabinet 29 near refrigerated compartment40 which minimizes solution precipitation, particularly that of osmiumtetroxide. Cap 120 is positioned slightly above the horizontal cabinetwall 123 which provides a relatively large unobstructed work surface tothe side of processing chamber 90. The chamber should be inert to allsolutions and has been made of Teflon plastic. Stainless steel and othermaterials may be used. As shown in FIG. 8, a small vent hole 124 isprovided on the upper inner wall of the processing chamber which allowsthe solutions to drain and also allows the fumes from solutions to beexhausted through an outside hole 125 by an exhaust fam connectedthrough a flexible conduit 131 to a suitable hood, not shown. That is,any fumes escaping through holes 124, 125 are drawn into the interior ofcabinet 29 and then exhausted by fan 130. Also, when cap 120 is removedany fumes escaping from chamber 90 are drawn into apertures 126 in topwall 123 as shown in dotted lines in FIG. 1. The fumes are then drawninside cabinet 29 and exhausted by fan 130. During processing thecapsules are secured in a tissue capsule rack of the type described inUS. Pat. No. 3,576,176, indicated at 92, FIG. 8.

The processing of tissue particles according to the invention has beendescribed as generally involving placing the tissue particles in thereceptacles; placing the receptacles in the receptacle rack, placing therack in the chamber and then drawing the solutions into the processingchamber in some predetermined time sequence. In this regard the presentinvention elminates level sensing in chamber 90 and allows the amount ofeach fluid drawn into the processing chamber to be precisely meteredaccording to the number of receptacles being processed. Thus, the panelboard in FIGS. 5 and 10 will be seen to have a volume control switch 132marked A, B, C and D which correspond in one embodiment to respectiveloads of one receptacle, (lOmL), two to four receptacles (25 ml.), fiveto 28 receptacles (50ml.) and 29 to 48 receptacles (100 ml.). Switchpositions A, B, C and D effectively select a corresponding micro-switchfrom the group of pump microswitches 115, 116, 117 and 118 and therebycontrol the level of fluid used of a particular solution. Additionalprogram circuitry, not shown, commands the pump to otherwise start andstop as required.

The circuitry also includes what is called a cycle control which isshown at 135 in FIGS. 5 and 10 and which allows either 1, 2 or 3solution changes of each stock solution. That is, the group of ten,three position switches shown in FIG. 5 and 10 at 135 are suitablyconnected into the illustrated program control circuitry so that eachsolution can be controlled during a particular program as to the numberof changes I, 2 or 3 of that solution. Thus, the cycle switch forsolution 1 may be set to call for solution I to have one change, thecycle switch for solution 2 may be set to call for solution 2 to havetwo changes, the cycle switch for solution 3 may be set to call forsolution 3 to have one change and so forth, as shown in FIG. 10.Furthermore, cycle switches may be set one way for one program andimmediately after that program is terminated the switches may be movedto a different array for the next program. In this manner, a versatilitynot heretofore achieved is provided. Those skilled in the art will, ofcourse, understand that the cycle switches by their settings controlwhatever program circuitry is employed. FIG. 10 is intended to simplyillustrate one such configuration based on using a stepping switch andstandard logic type circuits.

In another aspect of the programming, the present inention also providesfor a so-called time control." The time control operates on the basisthat each program is made up of some multiple of a standard base time,e.g., 15 minutes. A switch 150, shown in FIGS. 5 and 10, is a multipleposition timing switch and each position corresponds to a portion of thebase time period of 15 minutes. That is, switch can beset for any timefrom zero to 15 minutes. In addition to switch 150 the time control alsoincludes for each of the 10 solutions a multi-position time switchindicated at 155, which can be moved to a position to indicate eitherone, two, three, four, five, six etc., base units of time. Thus, if thetiming switch 150 is set on ten minutes and the particular time switchfor solution 1 is set on the 2 position this would mean that solution 1would stay in processing chamber 90 for 20 minutes (two times the base10 unit) and then run out. That is, each cycle with solution 1 would befor 20 minutes. If the corresponding cycle control switch 135 forsolution 1 were set on its 1 position the exchange of the solution Iwith the particles would only take place once but if, for example, thecycle control switch 135 were set on, say, its 2 position the exchangewould be repeated for another twenty minute period. The base timecontrol circuitry as well as the cycle control circuitry is, of course,connected to suitable programming circuitry as generally illustrated inFIG. 10. Thus, any solution can be regulated independently of all othersolutions as to the number of cycles and the time per cycle merely bypositioning the switches 135, 150 and 155.

The circuitry of the invention also incorporates a set of ten, threeposition, hold-pass switches 170 having positions indicated by N fornormal, P for pass and S for stop. Switches 170 are suitably connectedinto the program circuitry as indicated in FIG. 10. As backgroundinformation it may be noted that tissue processing of electronmicroscopy size tissue is subject at times to many collateral operationsfor experimental purposes or to get certain results supplementary tothose achieved by routine processing. That is, insome cases a programcan be set up which simply calls for the tissue particles to beprocessed step by step through all the solutions. In other cases howeverthe research investigator may, for example, want to bypass certainsolutions or he may want to, at least temporarily, terminate the programafter a particular solution. When the particular hold-pass switch 170 isset on N for normal the solution is used in a normal way and the switchposition has no effect on the program. However, when a selectedhold-pass switch 170 is set on P this switch setting directs the programcircuitry to pass the respective solution in the program and not use it.Thus, if the hold-pass switch 170 is set on P for solution 3, thissolution would not be used at all. Also,if the particular hold-passswitch 170 is set on S the associated program circuitry is so arrangedas to call for the program to shut down immediately after such solutionhas been dumped. Thus, if the operator technician is directed to stainall the particles in a special stain solution between, say, solutions 6and 7 the technician may set the hold-pass switch 170 corresponding tosolution 6 to the S position and the program will temporarily stop aftersolution 6 has been dumped to allow this collateral staining operation.g I

Considering further the circuitry and control panel shown in FIGS. and10, there is also incorporated a selector switch 160 having tenpositions, corresponding to the ten solutions, and which allows theoperator of the processor to select as a starting point in any programany of the ten respective solutions. Thus, a particular tissue mayrequire special processing, in only, say,

solutions 7, 8, 9 and 10 and this can be immediately ob- Y taineddependent only on a proper initial setting switch 160.

To complete the general explanation of the circuit it will be understoodthat given the concept of providing switch means to vary the base time,the cycles per solution, the particular solution starting point, thetime per cycle per solution and processing chamber level that many andvaried logic, stepping switches and other program and timingarrangements other than that shown generally and substantially in FIG.10 may be employed in conjunction with such switch means. That is, the10 cycle switches 135, the 10 hold-pass switches 170, the 10 timeswitches 155, the selector switch 160, the volume switch 132 and thebase time switch 150 may be used to control a variety of programcircuits suitable to the invention and well known in the art ofprogramming. The circuitry also includes a start switch 180 whosepurpose, and circuit arrangement, is to start a selected program oncethe various program selected switches have been positioned as requiredfor such program. In addition there is provided a release switch 181 anda so-called clean switch 182. The purpose of release switch 181 is toprovide a means to operate the dump valve so that in the event of amulfunction or for other reasons it becomes necessary to immediatelydrain chamber this can be done by actuating release switch 181. Switch181 is wired accordingly to override all other program commands. Theclean switch 182 serves another useful purpose and is wired in thecircuitry so as to command only certain solution control valves 73 tooperate in some predetermined sequence designed to flush or clean outthe chamber 90, manifold 75 and the various passages at the end of oneprogram and before another program starts. Thus, clean switch 182effectively commands a subsidiary program intended for cleaning thesystem and not for processing as such. In one embodiment clean switch182 and the associated circuitry are programmed to dump fluid fromchamber 90, then to rinse twice with propylene oxide, solution 9, thenonce with absolute alcohol, solution 8, then to dump and dry. Indicatorlamps 95, 96 and 97 indicate as shown in FIG. 10. Lamp 94 indicates awaste tank full condition.

It may be mentioned that an alternate manifold arrangement has beenemployed experimentally with level sensing as distinct from positivemetering. In the alternate arrangement the manifold allowed thesolutions to flow by gravity and entry was made through a single portlocated at the top of the processing chamber, however, this was foundnot to offer the advantages of the present invention. The illustratedmanifold of the'invention has proven particularly advantageous inproviding a means for shortening flow passages and for minimizingintermixing of solutions. It also provides a means for mounting thesolution control valves, the dump valve and the processing chamber valveas well as the metering pump. The use of a single solution entry anddischarge port in the processing chamber reduces possible points ofcontamination from l0 to 1. Thus, while the method and apparatus of ourown prior art worked and provided a highly significant advance in theart it can be seen that the present manifold arrangement has furtheradvanced the art and in novel and significant respects.

Agitation and vibration of the solutions during processing to increasesolution penetration through the receptacle pores has been tried but isgenerally not deemed necessary. Also, the required agitation orvibration apparatus has not been found to be justified by the resultsobtained. However, it is, of course, recognized that vibration oragitation may be employed and may in the future, as automatic tissueprocessing knowledge becomes more fully developed,prove justifiable incertain tissue techniques. Thus, a vibrator V, FIG. 8, may be employedto vibrate chamber 90 or a motor M, FIG. 8, may be employed to rotaterack 92 during processing somewhat like an electric ice cream freezerdasher to obtain solution agitation.

While not shown, the control panel and circuitry may include, dependingon the specific program circuitry employed, a set switch to set theprogram logic circuitry once the external control switches have beenselectively manually positioned and a reset switch to restore such logiccircuitry to a particular program state. Such set and reset switcheshave been employed in one experimental embodiment and a startingsequence used which used the solution indicator lamps shown in FIG. 10to indicate to the operator that a selected program has been set and isready to start on a particular solution indicated by the associatedsolution indicator lamp, in solution indicator lamp bank 140.

What is deemed most significant and most important to providing areliable day-to-day automatic electron microscopy processor is theachievement in this invention of a practical system of measuring ndminimizing fluid contamination. Also, of special importance to thepresent invention has been the achievement of a manual switch selectorsystem that readily adapts to any of many well known forms of programcircuitry to allow selection of base time, number of solution cycles,starting solutions, positive level measuring, particular solutions forparticular processing and cleaning programs, and alternate terminalsolutions for particular programs. The research investigator andhistology technician is thus provided with a new-apparatus and methodfor both duplicating hand processing procedures and, more importantly,for allowing completely new processing techniques not heretofore knownin the art.

While primarily directed to electron microscopy size tissue, it isrecognized that much of the fluid exchange and timing apparatus of theinvention lends itself to automatic slide tissue staining as welll as tolight microscopy processing directed to processing 3 to 5 millimeterthick pieces of tissue. For staining slides the processing chamber ofthe invention may be in the form of a rectangular well and used inconjunction with a conventional staining slide tray adapted to hold, forexample, 60 3 X 2 slides. The staining fluids would be recirculatedrather than dumped but would be periodically changed depending on theamount of use. The solenoid valves and pump operation could becontrolled accordingly and such staining could be performed at roomtemperature. For light microscopy work larger solution containers shouldbe employed and all lines and pump surfaces handling paraffin should bemaintained at a temperature high enough to keep the paraffin in a fluidstate. The tissue receptacles for such ligth microscopy processing couldbe of the type shown in U.S. Pat. No. 3,411,185 and a plurality of suchreceptacles could be stacked in a perforated basket for moving into andout of the processing chamber. The processing chamber could be the samerectangular well form used for staining thus adding to the versatility.As with staining the fluids would normally be recirculated and selectedfluids periodically changed depending on the amount of use.

I claim:

1. A closed processing system enabling a plurality of individualparticles of minute electron microscopy size, having at least onedimension greater than 1 millimeter, to be separately contained whilebeing bathed simultaneously for varying lengths of time in successiveselected aqueous and non-aqueous electron microscopy tissue solutions soas to fix, dehydrate and clear the particles preparatory to embedding,comprising:

a. a pluraltiy of uniform porous tissue receptacles each being adaptedto contain and physically isolate a group of said particles having atleast one particle per group, each receptacle having a body portiondefining an open cavity adapted to receive and retain a group ofparticles and closure means removably received by said body to enclosesaid cavity, the material forming said receptacles being inert to all ofsaid solutions and having for each of said solutions at least someporous portion permeable thereto;

b. cabinet means providing a housing and respective refrigerated andnon-refrigerated compartments therein;

c. a plurality of closed solution containers selectively mounted in saidrefrigerated and non-refrigerated compartments and each containing aparticular electron microscopy tissue processing solution andcollectively containing all of said aqueous and nonaqueous solutions;

d. a discharge pipe and an associated remotely and electricallycontrollable valve for each container connected to provide an individualselectively controllable flow path for each of said solutions to oneside of its'respective corresponding valve, selected said pipes havingat least a portion thereof positioned so as to be cooled in saidrefrigerated compartment;

e. a manifold structure fixedly mounted in said housing adjacent saidcompartments and mounting said container valves, said manifold structureproviding about the periphery thereof an inlet port for andcommunicating with an opposite side of each respective said containervalve, a centrally disposed, vertically directed passageway extendingthrough said manifold, each said inlet port being communicated to saidvertically directed passageway by a serparate one of a plurality ofradially extending passageways, a pump port located in said peripheryand a further radial passageway communicating said pump port with thevertical passageway;

f. a processing chamber mounted in said housing adjacent and above saidmanifold structure, said chamber having a removable top cover and beingadapted to receive and process selected numbers of said receptaclessimultaneously, said chamber having an entry-discharge port at thebottom thereof and a remotely and electrically controllable valvelocated between and controlling communication between saidentry-discharge port and the top end of said vertical passageway;

g. remotely controllable metering pump means and positioned within saidhousing adjacent said manifold structure and having a pump inlet communicating with said manifold structure pump port whereby said pump isenabled to withdraw and discharge through said pump port a positivelymeasured volume of a selected said solution;

h. waste solution collection means including a waste discharge remotelyelectrically controllable valve communicating on one side with thebottom end of said vertical passageway and on an opposite side with awaste solution collector whereby when said waste valve is opened allsolution fluids in said manifold passageways may flow to said wastecollector for disposal; and

i. remotely operable electrical control means for remotely operatingsaid container, chamber and waste valves and said pump means in apredetermined time sequence program whereby with a selected number ofsaid receptacles installed in said chamber and said cover on saidchamber said solutions are successively, selectively and independentlydrawn, externally measured, and admitted to said chamber in apredetermined volume, retained for predetermined times and drained fromsaid chamber in a corresponding processing sequence such that selectedofsaid aqueous and nonaqueous solutions are exchanged with each said groupof particles by penetration and drainage through said pores whilemaintaining said system closed and each said group physically isolated.

2. A system as claimed in claim 1 wherein said control means includeadjustable timing means enabling said predetermined time of retention tobe changed both with respect to all as well as selected ones of saidsolutions.

3. A system as claimed in claim 1 wherein said control means includesadjustable cycle switching means enabling selected solutions to becycled more than once through said chamber.

4. A system as claimed in claim 1 wherein said control means includestime unit switching means enabling said predetermined time of retentionto be varied with respect to selected said solutions.

5. A system as claimed in claim 1 wherein said control means includeshold-pass switching means enabling selected said solutions in one modeof switching to be by-passed and in another mode of switching toconstitute a program terminal stage.

6. A system as claimed in claim 1 wherein said control means includeslevel selection switching means enabling a plural selection of volumespumped into said chamber by said pump means.

7. A system as claimed in claim 1 wherein said control means includesswitching means enabling any said solution to be a beginning stage inthe said program.

8. A system as claimed in claim 1 wherein said control meansincludesprogram switching means adapted upon actuation to select certainsaid solutions and valves in a coordinated sequence adapted to flushselected said manifold and chamber surfaces for cleaning purposes.

9. A system as claimed in claim 1 wherein said manifold structurecomprises a vertically disposed substantially cylindrical blockcontaining said ports and passageways and having said container valvesand pump means mounted on the side thereof, said chamber valve beingmounted on an upper face and said waste valve on a lower face thereof. Ii

10. A system as claimed in claim 1 having fan exhaust and fumecollection means adapted to allow during operation of said systemcontinuous withdrawal of solution fumes both from within said housingand from the area surrounding the top of said chamber.

11. A system as claimed in claim 1 wherein said control means includes:

a. adjustable timing means enabling said predetermined times ofretention to be changed with respect to all said solutions,

b. adjustable cycle switching means enabling selected solutions to becycled more than once through said chamber,

c. time unit switching means enabling said predetermined time ofretention to be varied with respect to selected said solutions,

(1. hold-pass switching means enabling selected said solutions in onemode of switching to be by-passed and in another mode of switching toconstitute a program terminal stage,

e. level selection switching means enabling a plural selection ofvolumes to be pumped into said chamber by said pump means,

f. switching means enabling any said solution to be a beginning stage ina program, and

g. switching means adapted upon actuation to select certain saidsolutions and valves in a coordinate subprogram sequence adapted toflush selected said manifold and chamber surfaces for cleaning purposes.

12. A system as claimed in claim 1 including means to vibrate saidreceptacle during processing.

13. A system as claimed in claim 1 including means to agitate theselected said solution in said chamber during processing.

14. A system as claimed in claim 1 wherein said chamber is mounted insaid housing immediately adjacent said refrigerated compartment.

15. A system as claimed in claim 1 wherein said containers, pipes,valves, manifold structure, chamber and pump means are stationary withinsaid housing.

16. A processing system for electron microscopy, light microscopy,staining and similar tissue processing enabling a plurality ofindividual tissue specimens to be bathed simultaneously for varyinglengths of time in successive tissue solutions preparatory to otherprocedures, comprising:

a. holder means adapted to contain and physically isolate a plurality ofsaid specimens from each other, the material forming said holder meansbeing inert to all of said solutions and having for each of saidsolutions at least some portion permeable thereto;

b. cabinet means providing a housing and respective compartmentstherein;

c. a plurality of closed solution containers mounted in said housing andeach containing a particular tis sue processing solution;

d. a discharge pipe and an associated remotely and electricallycontrollable valve for each container connected to provide an individualselectively controllable flow path for each of said solutions to oneside of its respective corresponding valve;

e. a manifold structure fixedly mounted in said housing adjacent saidcompartments and mounting said container valves, said manifold structureproviding about the periphery thereof an inlet port for andcommunicating with an opposite side of each respective said containervalve, a centrally disposed, vertically directed passageway extendingthrough said manifold, each said inlet port being communicated to saidvertically directed passageway by a separate one of a plurality ofradially extending passageways, a pump port located in said peripheryand a further radial passageway communicating said pump port with thevertical passageway;

f. a processing chamber mounted in said housing adjacent and above saidmanifold structure, said chamber having a removable top cover and beingadapted to receive said holder means and to process selected numbers ofsaid specimens simultaneously, said chamber having an entry-dischargeport at the bottom thereof and a remotely and electrically controllablevalve located between and controlling communication between saidentrydischarge port and the top end of said vertical passageway;

g. remotely controllable metering pump means positioned within saidhousing adjacent said manifold structure and having a pump inletcommunicating with said manifold structure pump port whereby said pumpis enabled to withdraw and discharge through said pump port a positivelymeasured volume of a selected said solution; and

h. remotely operable electrical control means for remotely operatingsaid container and chamber valves and said pump means in a predeterminedtime sequence program whereby with a selected number of said specimensinstalled in said chamber and said cover on said chamber said solutionsare successively, selectively and independently drawn, externallymeasured, and admitted to said chamber in a predetrmined volume,retained for predetermined times and drained from said chamber throughvalvular means connected to said vertical passageway in a correspondingprocessing sequence such that selected of said solutions are exchangedwith each said specimen while maintaining said system closed and eachsaid specimen physically isolated from the other.

17. A system as claimed in claim 16 wherein said manifold structurecomprises a vertically disposed substantially cylindrical blockcontaining said ports and passageways and having said container valvesand pump means mounted on the side thereof.

18. A system as claimed in claim 17 having fan exhaust and fumecollection means adapted to allow during operation of said systemcontinuous withdrawal of solution fumes both from within said housingand from the area surrounding the top of said chamber.

Patent No. 3 l7l ,490 Dated November 13 1973 Inventor(s) Thomas D.Kinney and John E. -P. Pickett 7 It is certified that error appears inthe above-identified patent and that said Letters Patent are herebycorrected as shown below:

Col. line 41., "3,536,203" should be 3,526,203-.

Col. line 27, "switching" should be -switch-.

Col. line 64, "fam" should be fan-.

line 53; "ligth" should be -light.

Col. 6, line 54, "inention" should be -invention Col. 9, I l

Col. line 65, no should be inserted after "dimension" Col. 10, line 4,"plureltiy" should be -plurality-. Q Col. 10, line 56, "and" after"means" should be deleted.

Col. 14, line 4, "predetrmined" should be -predetermined-,

Signed and sealed this 6th day of August 1974.

I. (SEAL) Attest: I MCCOY M. GIBSON, JR. c. MARSHALL DANN AttestingOfficer Commissioner of Patents FORM PO-IOSO (10-69) USCOMM-Dc 60376-P69U45 GOVERNMENT PRINTING OFFICE: "i9 O-Ji6-33l,

1. A closed processing system enabling a plurality of individualparticles of minute electron microscopy size, having at least onedimension greater than 1 millimeter, to be separately contained whilebeing bathed simultaneously for varying lengths of time in successiveselected aqueous and non-aqueous electron microscopy tissue solutions soas to fix, dehydrate and clear the particles preparatory to embedding,comprising: a. a pluraltiy of uniform porous tissue receptacles eachbeing adapted to contain and physically isolate a group of saidparticles having at least one particle per group, each receptacle havinga body portion defining an open cavity adapted to receive and retain agroup of particles and closure means removably received by said body toenclose said cavity, the material forming said receptacles being inertto all of said solutions and having for each of said solutions at leastsome porous portion permeable thereto; b. cabinet means providing ahousing and respective refrigerated and non-refrigerated compartmentstherein; c. a plurality of closed solution containers selectivelymounted in said refrigerated and non-refrigerated compartments and eachcontaining a particular electron microscopy tissue processing solutionand collectively containing all of said aqueous and non-aqueoussolutions; d. a discharge pipe and an associated remotely andelectrically controllable valve for each container connected to providean individual selectively controllable flow path for each of saidsolutions to one side of its respective corresponding valve, selectedsaid pipes having at least a portion thereof positioned so as to becooled in said refrigerated compartment; e. a manifold structure fixedlymounted in said housing adjacent said compartments and mounting saidcontainer valves, said manifold structure providing about the peripherythereof an inlet port for and communicating with an opposite side ofeach respective said container valve, a centrally disposed, verticallydirected passageway extending through said manifold, each said inletport being communicated to said vertically directed passageway by aserparate one of a plurality of radially extending passageways, a pumpport located in said periphery and a further radial passagewaycommunicating said pump port with the vertical passageway; f. aprocessing chamber mounted in said housing adjacent and above saidmanifold structure, said chamber having a removable top cover and beingadapted to receive and process selected numbers of said receptaclessimultaneously, said chamber having an eNtry-discharge port at thebottom thereof and a remotely and electrically controllable valvelocated between and controlling communication between saidentry-discharge port and the top end of said vertical passageway; g.remotely controllable metering pump means and positioned within saidhousing adjacent said manifold structure and having a pump inletcommunicating with said manifold structure pump port whereby said pumpis enabled to withdraw and discharge through said pump port a positivelymeasured volume of a selected said solution; h. waste solutioncollection means including a waste discharge remotely electricallycontrollable valve communicating on one side with the bottom end of saidvertical passageway and on an opposite side with a waste solutioncollector whereby when said waste valve is opened all solution fluids insaid manifold passageways may flow to said waste collector for disposal;and i. remotely operable electrical control means for remotely operatingsaid container, chamber and waste valves and said pump means in apredetermined time sequence program whereby with a selected number ofsaid receptacles installed in said chamber and said cover on saidchamber said solutions are successively, selectively and independentlydrawn, externally measured, and admitted to said chamber in apredetermined volume, retained for predetermined times and drained fromsaid chamber in a corresponding processing sequence such that selectedof said aqueous and non-aqueous solutions are exchanged with each saidgroup of particles by penetration and drainage through said pores whilemaintaining said system closed and each said group physically isolated.2. A system as claimed in claim 1 wherein said control means includeadjustable timing means enabling said predetermined time of retention tobe changed both with respect to all as well as selected ones of saidsolutions.
 3. A system as claimed in claim 1 wherein said control meansincludes adjustable cycle switching means enabling selected solutions tobe cycled more than once through said chamber.
 4. A system as claimed inclaim 1 wherein said control means includes time unit switching meansenabling said predetermined time of retention to be varied with respectto selected said solutions.
 5. A system as claimed in claim 1 whereinsaid control means includes hold-pass switching means enabling selectedsaid solutions in one mode of switching to be by-passed and in anothermode of switching to constitute a program terminal stage.
 6. A system asclaimed in claim 1 wherein said control means includes level selectionswitching means enabling a plural selection of volumes pumped into saidchamber by said pump means.
 7. A system as claimed in claim 1 whereinsaid control means includes switching means enabling any said solutionto be a beginning stage in the said program.
 8. A system as claimed inclaim 1 wherein said control means includes program switching meansadapted upon actuation to select certain said solutions and valves in acoordinated sequence adapted to flush selected said manifold and chambersurfaces for cleaning purposes.
 9. A system as claimed in claim 1wherein said manifold structure comprises a vertically disposedsubstantially cylindrical block containing said ports and passagewaysand having said container valves and pump means mounted on the sidethereof, said chamber valve being mounted on an upper face and saidwaste valve on a lower face thereof.
 10. A system as claimed in claim 1having fan exhaust and fume collection means adapted to allow duringoperation of said system continuous withdrawal of solution fumes bothfrom within said housing and from the area surrounding the top of saidchamber.
 11. A system as claimed in claim 1 wherein said control meansincludes: a. adjustable timing means enabling said predetermined timesof retention to be changed with respect to all said solutions, b.adjustable cycle switching means enabling selected solutions to becycled more than once through said chamber, c. time unit switching meansenabling said predetermined time of retention to be varied with respectto selected said solutions, d. hold-pass switching means enablingselected said solutions in one mode of switching to be by-passed and inanother mode of switching to constitute a program terminal stage, e.level selection switching means enabling a plural selection of volumesto be pumped into said chamber by said pump means, f. switching meansenabling any said solution to be a beginning stage in a program, and g.switching means adapted upon actuation to select certain said solutionsand valves in a coordinate subprogram sequence adapted to flush selectedsaid manifold and chamber surfaces for cleaning purposes.
 12. A systemas claimed in claim 1 including means to vibrate said receptacle duringprocessing.
 13. A system as claimed in claim 1 including means toagitate the selected said solution in said chamber during processing.14. A system as claimed in claim 1 wherein said chamber is mounted insaid housing immediately adjacent said refrigerated compartment.
 15. Asystem as claimed in claim 1 wherein said containers, pipes, valves,manifold structure, chamber and pump means are stationary within saidhousing.
 16. A processing system for electron microscopy, lightmicroscopy, staining and similar tissue processing enabling a pluralityof individual tissue specimens to be bathed simultaneously for varyinglengths of time in successive tissue solutions preparatory to otherprocedures, comprising: a. holder means adapted to contain andphysically isolate a plurality of said specimens from each other, thematerial forming said holder means being inert to all of said solutionsand having for each of said solutions at least some portion permeablethereto; b. cabinet means providing a housing and respectivecompartments therein; c. a plurality of closed solution containersmounted in said housing and each containing a particular tissueprocessing solution; d. a discharge pipe and an associated remotely andelectrically controllable valve for each container connected to providean individual selectively controllable flow path for each of saidsolutions to one side of its respective corresponding valve; e. amanifold structure fixedly mounted in said housing adjacent saidcompartments and mounting said container valves, said manifold structureproviding about the periphery thereof an inlet port for andcommunicating with an opposite side of each respective said containervalve, a centrally disposed, vertically directed passageway extendingthrough said manifold, each said inlet port being communicated to saidvertically directed passageway by a separate one of a plurality ofradially extending passageways, a pump port located in said peripheryand a further radial passageway communicating said pump port with thevertical passageway; f. a processing chamber mounted in said housingadjacent and above said manifold structure, said chamber having aremovable top cover and being adapted to receive said holder means andto process selected numbers of said specimens simultaneously, saidchamber having an entry-discharge port at the bottom thereof and aremotely and electrically controllable valve located between andcontrolling communication between said entry-discharge port and the topend of said vertical passageway; g. remotely controllable metering pumpmeans positioned within said housing adjacent said manifold structureand having a pump inlet communicating with said manifold structure pumpport whereby said pump is enabled to withdraw and discharge through saidpump port a positively measured volume of a selected said solution; andh. remotely operable electrical control means for re-motely operatingsaid container and chamber valves and said pump means in a predeterminedtime sequence program whereby with a selected number of said specimensinstaLled in said chamber and said cover on said chamber said solutionsare successively, selectively and independently drawn, externallymeasured, and admitted to said chamber in a predetrmined volume,retained for predetermined times and drained from said chamber throughvalvular means connected to said vertical passageway in a correspondingprocessing sequence such that selected of said solutions are exchangedwith each said specimen while maintaining said system closed and eachsaid specimen physically isolated from the other.
 17. A system asclaimed in claim 16 wherein said manifold structure comprises avertically disposed substantially cylindrical block containing saidports and passageways and having said container valves and pump meansmounted on the side thereof.
 18. A system as claimed in claim 17 havingfan exhaust and fume collection means adapted to allow during operationof said system continuous withdrawal of solution fumes both from withinsaid housing and from the area surrounding the top of said chamber.